Genistein alleviates doxorubicin-induced cardiomyocyte autophagy and apoptosis via ERK/STAT3/c-Myc signaling pathway in rat model.

Doxorubicin (Dox) is a highly effective anti-neoplastic agent. Still, its utility in the clinic has been hindered by toxicities, including vomiting, hematopoietic suppression and nausea, with cardiotoxicity being the most serious side effect. Genistein (Gen) is a natural product with extensive biological effects, including anti-oxidation, anti-tumor, and cardiovascular protection. This study evaluated whether Gen protected the heart from Dox-induced cardiotoxicity and explored the underlying mechanisms. Male Sprague-Dawley (SD) rats were categorized into control (Ctrl), genistein (Gen), doxorubicin (Dox), genistein 20 mg/kg/day + doxorubicin (Gen20 + Dox) and genistein 40 mg/kg/day + doxorubicin (Gen40 + Dox) groups. Six weeks after injection, immunohistochemistry (IHC), transmission electron microscopy (TEM), and clinical cardiac function analyses were performed to evaluate the effects of Dox on cardiac function and structural alterations. Furthermore, each heart histopathological lesions were given a score of 0-3 in compliance with the articles for statistical analysis. In addition, molecular and cellular response of H9c2 cells toward Dox were evaluated through western blotting, Cell Counting Kit-8 (CCK8), AO staining and calcein AM/PI assay. Dox (5 µM in vitro and 18 mg/kg in vivo) was used in this study. In vivo, low-dose Gen pretreatment protected the rat against Dox-induced cardiac dysfunction and pathological remodeling. Gen inhibited extracellular signal-regulated kinase1/2 (ERK1/2)'s phosphorylation, increased the protein levels of STAT3 and c-Myc, and decreased the autophagy and apoptosis of cardiomyocytes. U0126, a MEK1/2 inhibitor, can mimic the effect of Gen in protecting against Dox-induced cytotoxicity both in vivo and in vitro. Molecular docking analysis showed that Gen forms a stable complex with ERK1/2. Gen protected the heart against Dox-induced cardiomyocyte autophagy and apoptosis through the ERK/STAT3/c-Myc signaling pathway.

Inhibition of MAPK/ERK pathway activation rescues congenital anomalies of the kidney and urinary tract (CAKUT) in Robo2PB/+ Gen1PB/+ mice.

Congenital anomalies of the kidney and urinary tract (CAKUT) have been attributed to genetic and environmental factors. However, monogenic and copy number variations cannot sufficiently explain the cause of the majority of CAKUT cases. Multiple genes through various modes of inheritance may lead to CAKUT pathogenesis. We previously showed that Robo2 and Gen1 coregulated the germination of ureteral buds (UB), significantly increasing CAKUT incidence. Furthermore, MAPK/ERK pathway activation is the central mechanism of these two genes. Thus, we explored the effect of the MAPK/ERK inhibitor U0126 in the CAKUT phenotype in Robo2PB/+Gen1PB/+ mice. Intraperitoneal injection of U0126 during pregnancy prevented the development of the CAKUT phenotype in Robo2PB/+Gen1PB/+ mice. Additionally, a single dose of 30 mg/kg U0126 on day 10.5 embryos (E10.5) was most effective for reducing CAKUT incidence and ectopic UB outgrowth in Robo2PB/+Gen1PB/+ mice. Furthermore, embryonic kidney mesenchymal levels of p-ERK were significantly decreased on day E11.5 after U0126 treatment, along with decreased cell proliferation index PHH3 and ETV5 expression. Collectively, Gen1 and Robo2 exacerbated the CAKUT phenotype in Robo2PB/+Gen1PB/+ mice through the MAPK/ERK pathway, increasing proliferation and ectopic UB outgrowth.

U0126 Compound Triggers Thermogenic Differentiation in Preadipocytes via ERK-AMPK Signaling Axis.

In recent years, thermogenic differentiation and activation in brown and white adipose tissues have been regarded as one of the major innovative and promising strategies for the treatment and amelioration of obesity. However, the pharmacological approach towards this process has had limited and insufficient commitments, which presents a greater challenge for obesity treatment. This research evaluates the effects of U0126 compound on the activation of thermogenic differentiation during adipogenesis. The results show that U0126 pretreatment primes both white and brown preadipocytes to upregulate thermogenic and mitochondrial genes as well as enhance functions during the differentiation process. We establish that U0126-mediated thermogenic differentiation induction occurs partially via AMPK activation signaling. The findings of this research suggest U0126 as a promising alternative ligand in pursuit of a pharmacological option to increase thermogenic adipocyte formation and improve energy expenditure. Thus it could pave the way for the discovery of therapeutic drugs for the treatment of obesity and its related complications.

Loss of PFKFB4 induces cell cycle arrest and glucose metabolism inhibition by inactivating MEK/ERK/c-Myc pathway in cervical cancer cells.

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) was reported to be necessary for tumour growth in several cancers. However, the function of PFKFB4 in cervical cancer has not been clearly elucidated. Bioinformatics analysis was applied to detect the expression of PFKFB4 in cervical cancer and the association with survival prognosis. The effect of PFKFB4 on cervical cancer cells growth, cycle, invasion, migration and glucose metabolism was investigated by loss-of-function approaches in vitro. The association between PFKFB4 and MEK/ERK/c-Myc pathway was identified by western blot assay. We found that PFKFB4 was highly expressed in cervical cancer samples and its overexpression led to a poor prognosis of cervical cancer patients. Knock down of PFKFB4 reduced cell growth, blocked cell cycle, inhibited cell invasion and migration, and blocked glucose metabolism in cervical cancer cells. Our findings afforded a theoretical basis for further research on the treatment of cervical cancer based on the control of PFKFB4 expression. Impact StatementWhat is already known on this subject? PFKFB4 was overexpressed in several kinds of cancers and its requirement for tumour growth has been confirmed in cancers such as glioma and breast cancer. However, the function of PFKFB4 in cervical cancer cells has not been clearly elucidated. A bioinformatics study showed that PFKFB4 was a member of a six-gene signature associated with glycolysis to predict the prognosis of patients with cervical cancer. However, the relationship between PFKFB4 and glucose metabolism in cervical cancer has not been revealed.What do the results of this study add? Our results showed that PFKFB4 was highly expressed in cervical cancer samples and its overexpression led to a poor prognosis of cervical cancer patients. Moreover, the administration of si-PFKFB4 significantly reduced cell growth ability, blocked cell cycle, restrained the mobility and suppressed the glucose metabolism in cervical cancer cells partially by inactivating MEK/ERK/c-Myc pathway.What are the implications of these findings for clinical practice and/or further research? Our findings afforded a theoretical basis for further research on the treatment of cervical cancer based on the control of PFKFB4 expression.

U0126 pretreatment inhibits cisplatin-induced apoptosis and autophagy in HEI-OC1 cells and cochlear hair cells.

Deafness is the most common sensory disorder in the world. Ototoxic drugs are common inducing factors of sensorineural hearing loss, and cochlear hair cell (HC) damage is the main concern of the present studies. Cisplatin is a widely used, highly effective antitumor drug, but some patients have experienced irreversible hearing loss as a result of its application. This hearing loss is closely related to HC apoptosis and autophagy. U0126 is a specific inhibitor of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling pathway and has neuroprotective effects. For example, the neuroprotective effect of U0126 on ischemic stroke has been widely recognized. In neural cells, U0126 can prevent death due to excess glutamate, dopamine, or zinc ions. However, no studies of U0126 and ototoxic drug-induced injury have been reported to date. In the present study, we found that U0126 pretreatment significantly reduced the apoptosis and autophagy of HCs in auditory House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear HCs. In addition, U0126 reduced the cisplatin-induced production of reactive oxygen species as well as the cisplatin-induced decrease in the mitochondrial membrane potential. These findings suggest that U0126 may be a potential therapeutic candidate for the prevention of cisplatin-induced ototoxicity.

ß-Arrestin2 deficiency attenuates oxidative stress in mouse hepatic fibrosis through modulation of NOX4.

Hepatic fibrosis is a disease characterized by excessive deposition of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is responsible for most of ECM production. Oxidative stress and reactive oxygen species (ROS) may be important factors leading to liver fibrosis. NADPH oxidase 4 (NOX4) is the main source of ROS in hepatic fibrosis, but the mechanism by which NOX4 regulates oxidative stress is not fully understood. ß-Arrestin2 is a multifunctional scaffold protein that regulates receptor endocytosis, signaling and trafficking. In this study, we investigated whether ß-arrestin2 regulated oxidative stress in hepatic fibrosis. Both ß-arrestin2 knockout (Arrb2 KO) mice and wild-type mice were intraperitoneally injected with carbon tetrachloride (CCl4) to induce hepatic fibrosis. Arrb2 KO mice showed significantly attenuated liver fibrosis, decreased ROS levels and NOX4 expression, and reduced collagen levels in their livers. In vitro, NOX4 knockdown significantly inhibited ROS production, and decreased expression of alpha-smooth muscle actin in angiotensin II-stimulated human HSC cell line LX-2. Through overexpression or depletion of ß-arrestin2 in LX-2 cells, we revealed that decreased ß-arrestin2 inhibited ROS levels and NOX4 expression, and reduced collagen production; it also inhibited activation of ERK and JNK signaling pathways. These results demonstrate that ß-arrestin2 deficiency protects against liver fibrosis by downregulating ROS production through NOX4. This effect appears to be mediated by ERK and JNK signaling pathways. Thus, targeted inhibition of ß-arrestin2 might reduce oxidative stress and inhibit the progression of liver fibrosis.

Methylene blue ameliorates brain edema in rats with experimental ischemic stroke via inhibiting aquaporin 4 expression.

Brain edema is a common and serious complication of ischemic stroke with limited effective treatment. We previously reported that methylene blue (MB) attenuated ischemic brain edema in rats, but the underlying mechanisms remained unknown. Aquaporin 4 (AQP4) in astrocytes plays a key role in brain edema. We also found that extracellular signal-regulated kinase 1/2 (ERK1/2) activation was involved in the regulation of AQP4 expression in astrocytes. In the present study, we investigated whether AQP4 and ERK1/2 were involved in the protective effect of MB against cerebral edema. Rats were subjected to transient middle cerebral artery occlusion (tMCAO), MB (3 mg/kg, for 30 min) was infused intravenously through the tail vein started immediately after reperfusion and again at 3 h after ischemia (1.5 mg/kg, for 15 min). Brain edema was determined by MRI at 0.5, 2.5, and 48 h after tMCAO. The decreases of apparent diffusion coefficient (ADC) values on diffusion-weighted MRI indicated cytotoxic brain edema, whereas the increase of T2 MRI values reflected vasogenic brain edema. We found that MB infusion significantly ameliorated cytotoxic brain edema at 2.5 and 48 h after tMCAO and decreased vasogenic brain edema at 48 h after tMCAO. In addition, MB infusion blocked the AQP4 increases and ERK1/2 activation in the cerebral cortex in ischemic penumbra at 48 h after tMCAO. In a cell swelling model established in cultured rat astrocyte exposed to glutamate (1 mM), we consistently found that MB (10 µM) attenuated cell swelling, AQP4 increases and ERK1/2 activation. Moreover, the ERK1/2 inhibitor U0126 (10 µM) had the similar effects as MB. These results demonstrate that MB improves brain edema and astrocyte swelling, which may be mediated by the inhibition of AQP4 expression via ERK1/2 pathway, suggesting that MB may be a potential choice for the treatment of brain edema.

IL-8 and MCP-1/CCL2 regulate proteolytic activity in triple negative inflammatory breast cancer a mechanism that might be modulated by Src and Erk1/2.

Inflammatory breast cancer (IBC) is a highly metastatic and lethal breast cancer. As many as 25-30% of IBCs are triple negative (TN) and associated with low survival rates and poor prognosis. We found that the microenvironment of IBC is characterized by high infiltration of tumor associated macrophages (TAMs) and by over-expression of the cysteine protease cathepsin B (CTSB). TAMs in IBC secrete high levels of the cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1/CCL2) compared to non-IBC patients. Herein, we tested the roles of IL-8 and MCP-1/CCL2 in modulating proteolytic activity and invasiveness of TN-non-IBC as compared to TN-IBC and addressed the underlying molecular mechanism(s) for both cytokines. Quantitative real time PCR results showed that IL-8 and MCP-1/CCL2 were significantly overexpressed in tissues of TN-IBCs. IL-8 and MCP-1/CCL2 induced CTSB expression and activity of the p-Src and p-Erk1/2 signaling pathways relevant for invasion and metastasis in TN-non-IBC, HCC70 cells and TN-IBC, SUM149 cells. Dasatinib, an inhibitor of p-Src, and U0126, an inhibitor of p-Erk1/2, down-regulated invasion and expression of CTSB by HCC70 and SUM149 cells, a mechanism that is reversed by IL-8 and MCP-1/CCL2. Our study shows that targeting the cytokines IL-8 and MCP-1/CCL2 and associated signaling molecules may represent a promising therapeutic strategy in TN-IBC patients.

Albumin induces CD44 expression in glomerular parietal epithelial cells by activating extracellular signal-regulated kinase 1/2 pathway.

De novo expression of CD44 in glomerular parietal epithelial cells (PECs) leads to a prosclerotic and migratory PEC phenotype in glomerulosclerosis. However, the regulatory mechanisms underlying CD44 expression by activated PECs remain largely unknown. This study was performed to examine the mediators responsible for CD44 induction in glomerular PECs in association with diabetes. CD44 expression and localization were evaluated in the glomeruli of Zucker diabetic rat kidneys and primary cultured PECs upon albumin stimulation. Real-time polymerase chain reaction confirmed an albuminuria-associated upregulation of the CD44 gene in the glomeruli of diabetic rats. Immunostaining analysis of diabetic kidneys further revealed an increase in CD44 in hypertrophic PECs, which often contain albumin-positive vesicles. Losartan treatment significantly attenuated albuminuria and lowered CD44 protein levels in the diabetic kidneys. In primary cultured rat PECs, rat serum albumin (0.25-1 mg/ml) caused a dose-dependent upregulation of CD44, claudin-1, and megalin protein expression, which was accompanied by an activation of extracellular signal-regulated kinase1/2 (ERK1/2) signaling. Albumin-induced CD44 and claudin-1 expression were greatly suppressed in the presence of the ERK1/2 inhibitor, U0126. In addition, knockdown of megalin by small interfering RNA interference in PECs resulted in a significant reduction of albumin-induced CD44 and claudin-1 proteins. Taken together, our results demonstrate that albumin induces CD44 expression by PECs via the activation of the ERK signaling pathway, which is partially mediated by endocytic receptor megalin.

Pre-clinical effects of highly potent MEK1/2 inhibitors on rat cerebral vasculature after organ culture and subarachnoid haemorrhage.

Background: Aneurysmal subarachnoid haemorrhage (SAH) is a variant of haemorrhagic stroke with a striking 50% mortality rate. In addition to the initial insult, secondary delayed brain injury may occur days after the initial ischemic insult and is associated with vasospasms leading to delayed cerebral ischemia. We have previously shown that the MEK1/2 inhibitor U0126 improves neurological assessment after SAH in rats. Aim: The purpose of the present study was to analyse the impact of a broad selection of high potency MEK1/2 inhibitors in an organ culture model and use the IC50 values obtained from the organ culture to select highly potent inhibitors for pre-clinical in vivo studies. Results: Nine highly potent mitogen activated protein kinase kinase (MEK1/2) inhibitors were screened and the two most potent inhibitors from the organ culture screening, trametinib and PD0325901, were tested in an in vivo experimental rat SAH model with intrathecal injections. Subsequently, the successful inhibitor trametinib was administered intraperitoneally in a second in vivo study. In both regimens, trametinib treatment caused significant reductions in the endothelin-1 induced contractility after SAH, which is believed to be associated with endothelin B receptor up-regulation. Trametinib treated rats showed improved neurological scores, evaluated by the ability to traverse a rotating pole, after induced SAH. Conclusion: The PD0325901 treatment did not improve the neurological score after SAH, nor showed any beneficial therapeutic effect on the contractility, contrasting with the reduction in neurological deficits seen after trametinib treatment. These data show that trametinib might be a potential candidate for treatment of SAH.

LongShengZhi Capsule reduces carrageenan-induced thrombosis by reducing activation of platelets and endothelial cells.

Formation of thrombosis is associated with activation of platelets and endothelial cells. The effect of LongShengZhi Capsule (LSZ), a traditional Chinese medicine used for treatment of vascular diseases, on thrombosis was investigated in this study. BALB/c mice were induced thrombosis by injection of carrageenan while receiving pre or simultaneous LSZ treatment. We also compared the therapeutic effects of LSZ and clopidogrel on formed thrombi. LSZ inhibited carrageenan-induced thrombi in mouse tissue vessels. In addition, LSZ but not clopidogrel reduced formed thrombi with a short time window. The reduction of thrombi by LSZ was associated with reduced serum P-selectin, reduced expression of TNF-α and P-selectin and activated matrix metalloproteinase 2 expression in tissues. In vitro, LSZ decreased thrombin-induced human platelet clot retraction which was associated with inactivation of AKT and ERK1/2. LSZ also reduced adhesion of platelets or THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein or lipopolysaccharide. The anti-adherent actions of LSZ was attributed to reduction of oxidative stress, expression of platelet receptors (P2Y12, PAR4 and CD36) and AKT activity in platelets. LSZ also reduced adhesion molecules or tissue factor but activated tissue factor pathway inhibitor expression in HUVECs. Taken together, our study demonstrates the antithrombotic properties of LSZ by reducing activation of platelets and endothelial cells, and suggests its potential application in clinics.

Differential effects of MEK inhibitors on rat neural stem cell differentiation: Repressive roles of MEK2 in neurogenesis and induction of astrocytogenesis by PD98059.

Neural stem cells (NSCs) proliferate and differentiate into neurons and glia depending on the culture environment. However, the underlying mechanisms determining the fate of NSCs are not fully understood. Growth factors facilitate NSC proliferation through mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and MAPK activation, and NSCs differentiate into neurons, astrocytes, or oligodendrocytes when mitogens are withdrawn from the culture media. Here, we aimed to identify the effects and roles of MEK signaling on the determination of NSC fate. MEK inhibitors, U0126, SL327, and PD98059, had differential effects on NSC differentiation. U0126 and SL327, which are known to inhibit MEK1 and MEK2, induced neuronal differentiation, whereas PD98059, which is reported to preferentially inhibit MEK1 at higher concentrations, increased astrocytogenesis. Knockdown of MEK2 using small interfering RNA increased neurogenesis and over-expression of wild type (WT) MEK2 inhibited neurogenesis, suggesting a repressive role of MEK2 in neuronal differentiation. The chemical structure of PD98059 appears to be important for induction of astrocytogenesis because not only PD98059 (2'-amino-3'-methoxyflavone) but also its chemical structural mimetic, 3'-methoxyflavone, enhanced astrocytogenesis. Therefore, in our study, we suggest that MEK inhibitors have distinct functions in determining NSC fate. Inhibition of MEK2 is important for induction of neurogenesis in NSCs. U0126 and SL327 increase neurogenesis through MEK2 inhibition, whereas PD98059 induced astrocytogenesis in NSCs, which is mediated by the chemical structure, particularly the 3'-methoxy group rather than its renowned MEK1 inhibition.

Relationship between Erk1/2 signal pathway and nerve cell apoptosis rats with ischemic stroke.

To investigate the relationship between the Erk1/2 signal pathway and neuronal apoptosis in ischemic stroke rats. Male SD(Sprague Dawley)  rats (n = 24) were randomly divided into three groups, each containing 8 rats: sham-operated group, MCAO(Midle cerebral artery oclusion)  group, and MCAO + U0126 intervention group (U0126 group). In in vitro trial, primary cortical nerve cells were divided into three groups: control group, OGD(Oxygen and glucose deprivation)  group, and U0126 intervention group (U0126 group). In vivo protein expression levels of Erk1/2, p-Erk1/2 and Bcl-2 were determined using western blot. The expressions of Bcl-2, Bcl-xl and Bax were assayed using immunohistochemical staining. Nerve cell mortality in cerebral tissue was detected using TUNEL staining. In in vitro trials, cell apoptosis was assayed with flow cytometry and LDH release. The activity of caspase-3 was determined. Nerve cell apoptosis was determined using Hoechst33258 staining method. In in vivo trial, it was found that the protein expression level of p-ERK1/2 in cerebral tissue in the MCAO group was significantly increased, when compared with that of the sham-operated group, while the protein expression level of p-Erk1/2 in the U0126 group was significantly lower than that in the MCAO group. The expression levels of Bcl-2 and Bcl-xl in the MCAO group were significantly lower than the corresponding expression levels in the sham-operated group, while the expressions of Bcl-2 and Bcl-xl in the U0126 group were significantly lower than those in MCAO group. In MCAO group, the expression of Bax was significantly higher than that in the sham-operated group, while Bax expression was higher in U0126 than in MCAO group. There were significantly higher number of dead nerve cells in MCAO group than in the sham-operated group, while nerve cell mortality in U0126 group was significantly lower than in MCAO group. In in vitro trials, flow cytometry revealed significantly higher apoptosis of OGD-treated nerve cells, relative to the control group. Nerve cells exposed to U0126 and treated with ODR (Oxygen-dependent repressor)  were significantly decreased in population, when compared with single OGD treatment group. The LDH release level of nerve cells treated OGD was significantly increased, when compared with that of the control group. However, LDH release level of nerve cells treated with OGD after U0126 intervention was significantly decreased, relative to the single OGD treatment group. The dilution of nerve cell nucleus after OGD treatment was significantly increased, when compared with that of the control group. For nerve cells treated with ODR after U0126 intervention, the nuclear dilution was significantly decreased, relative to that of nerve cell nucleus in the single OGD treatment group. The OGD treatment led to significant increase in nerve cell caspase-3 activity, relative the control group. However, the caspase-3 activity of nerve cells treated with ODR after U0126 intervention was significantly decreased, when compared with single OGD treatment group. The activation of Erk1/2 signal pathway during ischemic stroke promotes apoptosis of nerve cells. Based on these findings, it can be reasonably inferred that the ERK1/2 signal pathway may be an important target for treating ischemic stroke.

Irisin exerts a therapeutic effect against myocardial infarction via promoting angiogenesis.

Irisin, a myokine, is cleaved from the extracellular portion of fibronectin domain-containing 5 protein in skeletal muscle and myocardium and secreted into circulation as a hormone during exercise. Irisin has been found to exert protective effects against lung and heart injuries. However, whether irisin influences myocardial infarction (MI) remains unclear. In this study we investigated the therapeutic effects of irisin in an acute MI model and its underlying mechanisms. Adult C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery and treated with irisin for 2 weeks after MI. Cardiac function was assessed using echocardiography. We found that irisin administration significantly alleviated MI-induced cardiac dysfunction and ventricular dilation at 4 weeks post-MI. Irisin significantly reduced infarct size and fibrosis in post-MI hearts. Irisin administration significantly increased angiogenesis in the infarct border zone and decreased cardiomyocyte apoptosis, but did not influence cardiomyocyte proliferation. In human umbilical vein endothelial cells (HUVEC), irisin significantly increased the phosphorylation of ERK, and promoted the migration of HUVEC detected in wound-healing and transwell chamber migration assay. The effects of irisin were blocked by the ERK inhibitor U0126. In conclusion, irisin improves cardiac function and reduces infarct size in post-MI mouse heart. The therapeutic effect is associated with its pro-angiogenic function through activating ERK signaling pathway.

Astragaloside IV attenuates myocardial ischemia/reperfusion injury in rats via inhibition of calcium-sensing receptor-mediated apoptotic signaling pathways.

Astragaloside IV (AsIV) is an active saponin extracted from Astragalus membranaceus, which has shown cardioprotective effects in a number of experimental animals. In this study we investigated the molecular mechanisms by which AsIV attenuated the myocardial ischemia reperfusion (MI/R)-induced injury in vitro and in vivo by focusing on calcium-sensing receptor (CaSR) and extracellular signal-regulated kinase 1/2 (ERK1/2). Rat neonatal cardiac myocytes were subjected to a hypoxia/reoxygenation (H/R) procedure in vitro, which significantly decreased the cell viability, increased lactate dehydrogenase (LDH) release, induced cardiomyocyte apoptosis, and increased [Ca2+]i. H/R also increased the expression of CaSR and decreased ERK1/2 phosphorylation levels in H/R-exposed myocytes. Pretreatment with AsIV (60 µmol/L) significantly improved the cell viability and decreased LDH release, attenuated myocyte apoptosis, decreased [Ca2+]i and CaSR expression, and increased the ERK1/2 phosphorylation levels. The protective effects of AsIV against H/R injury were partially inhibited by co-treatment with a CaSR agonist, gadolinium chloride (GdCl3) or with a specific ERK1/2 inhibitor U0126. For in vivo studies, a rat MI/R model was established. Pre-administration of AsIV (80 mg/kg every day, ig) significantly decreased the myocardium infarct size, creatine kinase-MB (CK-MB) production, serum cardiac troponin (cTnI) levels, and cardiomyocyte apoptosis in the rats with MI/R injury. The therapeutic effects of AsIV were associated with the downregulation of CaSR expression and upregulation of ERK1/2 phosphorylation in myocardial tissues. In summary, astragaloside IV attenuates myocardial I/R injury via inhibition of CaSR/ERK1/2 and the related apoptotic signaling pathways.

The Regional Specific Alterations in BBB Permeability are Relevant to the Differential Responses of 67-kDa LR Expression in Endothelial Cells and Astrocytes Following Status Epilepticus.

Status epilepticus (a prolonged seizure activity, SE) differently affects vasogenic edema formation and dystrophin-aquaporin 4 (AQP4) expressions between the rat hippocampus and the piriform cortex (PC). In the present study, we explored whether the 67-kDa laminin receptor (LR) expression was relevant to the regional specific susceptibility of vasogenic edema at 3 days after SE. In spite of no difference in expression levels of 67-kDa LR, dystrophin, and AQP4 under physiological conditions, SE-induced serum extravasation was more severe in the PC than the hippocampus. Western blots demonstrated that SE reduced expression levels of 67-kDa LR, dystrophin, and AQP4 in the PC, but not in the hippocampus proper. Immunofluorescent studies revealed that SE increased 67-kDa LR expression in reactive CA1 astrocyte, but reduced it in the PC and the molecular layer of the dentate gyrus due to massive astroglial loss. Furthermore, SE decreased expressions of endothelial 67-kDa LR and SMI-71 (endothelial brain barrier antigen) in these regions. The 67-kDa LR neutralization evoked serum extravasation in these regions of normal animals without astroglial loss. Similar to SE, 67-kDa LR neutralization also reduced dystrophin-AQP4 expressions in the PC more than the total hippocampus. Furthermore, 67-kDa LR IgG infusion increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), but not c-Jun N-terminal kinase, independent of phosphoprotein enriched in astrocytes of 15 kDa (PEA15) activity. Co-treatment of U0126 (an ERK1/2 inhibitor) alleviated vasogenic edema formation and the reduced dystrophin-AQP4 expressions induced by 67-kDa LR neutralization. The 67-kDa LR IgG infusion also increased the susceptibility to SE induction. Therefore, our findings suggested that the cellular specific alterations in 67-kDa LR expression might be involved in the severity of SE-induced vasogenic edema formation in regional specific manners, which might affect the susceptibility to SE induction.

Induction of MEK/ERK activity by AZD8055 confers acquired resistance in neuroblastoma.

Mammalian target of rapamycin (mTOR) complex (mTORC) is frequently activated in diverse cancers. Although dual mTORC1/2 inhibitors are currently under development to treat various malignancies, the emergence of drug resistance has proven to be a major complication. AZD8055 is a novel, potent ATP-competitive and specific inhibitor of mTOR kinase activity, which blocks both mTORC1 and mTORC2 activation. In this study, we acquired AZD8055-resistant neuroblastoma (NB) cell sublines by using prolonged stepwise escalation of AZD8055 exposure (4-12 weeks). Here we demonstrate that the AZD8055-resistant sublines (TGW-R and SMS-KAN-R) exhibited marked resistance to AZD8055 compared to the parent cells (TGW and SMS-KAN). The cell cycle G1/S transition was advanced in resistant cells. In addition, the resistance against AZD8055 correlated with over-activation of MEK/ERK signaling pathway. Furthermore, combination of AZD8055 and MEK inhibitor U0126 enhanced the growth inhibition of resistant cells significantly in vitro and in vivo. In conclusion, these data show that targeting mTOR kinase and MEK/ERK signaling simultaneously might help to overcome AZD8055 resistance in NB.

Sulforaphane attenuates microglia-mediated neuronal necroptosis through down-regulation of MAPK/NF-κB signaling pathways in LPS-activated BV-2 microglia.

Sulforaphane (SFN), a natural dietary isothiocyanate in cruciferous vegetables such as broccoli and cabbage, has very strong anti-inflammatory activity. Activation of microglia leads to overexpression of a series of pro-inflammatory mediators, which play a vital role in neuronal damage. SFN may have neuroprotective effects in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying SFN's protection of neurons against microglia-mediated neuronal damage are not fully understood. Here, we investigated how SFN attenuated microglia-mediated neuronal damage. Our results showed that SFN could not directly protect the viability of neurons following pro-inflammatory mediators, but increased the viability of BV-2 microglia and down-regulated the mRNA and protein levels of pro-inflammatory mediators including TNF-α, IL-1ß, IL-6 and iNOS in a concentration-dependent manner in BV-2 cells. SFN also significantly blocked the phosphorylation of MAPKs (p38, JNK, and ERK1/2) and NF-κB p65, both by itself and with MAPK inhibitors (SB203580, SP 600125, and U0126) or an NF-κB inhibitor (PDTC). The expression of pro-inflammatory proteins was also blocked by SFN with or without inhibitors. Further, SFN indirectly increased the viability and maintained the morphology of neurons, and the protein expression of RIPK3 and MLKL was significantly suppressed by SFN in neuronal necroptosis through p38, JNK, and NF-κB p65 but not ERK1/2 signaling pathways. Together, our results demonstrate that SFN attenuates LPS-induced pro-inflammatory responses through down-regulation of MAPK/NF-κB signaling pathway in BV-2 microglia and thus indirectly suppresses microglia-mediated neuronal damage.

MAPKK Inhibitor U0126 Inhibits Plasmodiophora brassicae Development.

Mitogen-activated protein kinase (MAPK) cascades play a central role in cellular growth, proliferation, and survival. MAPK cascade genes have been extensively investigated in model plants, mammals, yeast, and fungi but are not characterized in Plasmodiophora brassicae, which causes clubroot disease in cruciferous plants. Here, we identified 7 PbMAPK, 3 PbMAPKK, and 9 PbMAPKKK genes in the P. brassicae genome. Transcriptional profiling analysis demonstrated that several MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK) genes were preferentially expressed in three different zoosporic stages. Based on yeast two-hybrid assays, PbMAKKK7 interacted with PbMAKK3 and PbMAKK3 interacted with PbMAK1/PbMAK3. The PbMAKKK7-PbMAKK3-PbMAK1/PbMAK3 cascade may be present in P. brassicae. U0126, a potent and specific inhibitor of MAPKK, could inhibit the germination of P. brassicae resting spores. U0126 was used to treat the resting spores of P. brassicae and coinoculate rapeseed, and was proven to significantly relieve the severity of clubroot symptoms in the host plant and delay the life cycle of P. brassicae. These results suggest that MAPK signaling pathways may play important roles in P. brassicae growth, development, and pathogenicity.

Effects of Sunitinib and Other Kinase Inhibitors on Cells Harboring a PDGFRB Mutation Associated with Infantile Myofibromatosis.

Infantile myofibromatosis represents one of the most common proliferative fibrous tumors of infancy and childhood. More effective treatment is needed for drug-resistant patients, and targeted therapy using specific protein kinase inhibitors could be a promising strategy. To date, several studies have confirmed a connection between the p.R561C mutation in gene encoding platelet-derived growth factor receptor beta (PDGFR-beta) and the development of infantile myofibromatosis. This study aimed to analyze the phosphorylation of important kinases in the NSTS-47 cell line derived from a tumor of a boy with infantile myofibromatosis who harbored the p.R561C mutation in PDGFR-beta. The second aim of this study was to investigate the effects of selected protein kinase inhibitors on cell signaling and the proliferative activity of NSTS-47 cells. We confirmed that this tumor cell line showed very high phosphorylation levels of PDGFR-beta, extracellular signal-regulated kinases (ERK) 1/2 and several other protein kinases. We also observed that PDGFR-beta phosphorylation in tumor cells is reduced by the receptor tyrosine kinase inhibitor sunitinib. In contrast, MAPK/ERK kinases (MEK) 1/2 and ERK1/2 kinases remained constitutively phosphorylated after treatment with sunitinib and other relevant protein kinase inhibitors. Our study showed that sunitinib is a very promising agent that affects the proliferation of tumor cells with a p.R561C mutation in PDGFR-beta.